SLAC-PUB-12101
September 2006
Sulfur K-Edge XAS and DFT Calculations on Nitrile
Hydratase: Geometric and Electronic Structure of the
Non-heme Iron Active Site
Abhishek Dey,† Marina Chow,† Kayoko Taniguchi,§ Priscilla Lugo-Mas,⊥
Steven Davin,⊥ Mizuo Maeda,§ Julie A. Kovacs,*,⊥ Masafumi Odaka,*,§
Keith O. Hodgson,*,†,‡ Britt Hedman,*,‡ and Edward I. Solomon*,†
Contribution from the Department of Chemistry, Stanford UniVersity, Stanford,
California 94305, Bioengineering Laboratory, RIKEN, Wako, Saitama 351-0198, Japan,
Department of Chemistry, UniVersity of Washington, Seattle, Washington 98195, and Stanford
Synchrotron Radiation Laboratory, SLAC, Stanford UniVersity, Stanford, California 94309
Received July 23, 2005; E-mail: [email protected]
Abstract: The geometric and electronic structure of the active site of the non-heme iron enzyme nitrile
hydratase (NHase) is studied using sulfur K-edge XAS and DFT calculations. Using thiolate (RS-)-, sulfenate
(RSO-)-, and sulfinate (RSO2-)-ligated model complexes to provide benchmark spectral parameters, the
results show that the S K-edge XAS is sensitive to the oxidation state of S-containing ligands and that the
spectrum of the RSO- species changes upon protonation as the S-O bond is elongated (by ∼0.1 Å).
These signature features are used to identify the three cysteine residues coordinated to the low-spin FeIII
in the active site of NHase as CysS-, CysSOH, and CysSO2- both in the NO-bound inactive form and in
the photolyzed active form. These results are correlated to geometry-optimized DFT calculations. The preedge region of the X-ray absorption spectrum is sensitive to the Zeff of the Fe and reveals that the Fe in
[FeNO]6 NHase species has a Zeff very similar to that of its photolyzed FeIII counterpart. DFT calculations
reveal that this results from the strong π back-bonding into the π* antibonding orbital of NO, which shifts
significant charge from the formally t26 low-spin metal to the coordinated NO.
Introduction
Nitrile hydratase (NHase) hydrolyzes nitriles to amides and
is used in the industrial preparation of acrylamide.1-3 The active
site can contain either a low-spin FeIII or a low-spin CoIII ion
coordinated by three cysteines and two deprotonated amides
(two amides and two cysteines occupying the equatorial plane
with an axial cysteine).5-7 The Fe enzyme is isolated in a
diamagnetic NO-bound form, activated by irradiation, and is
proposed to be stabilized by the presence of butyric acid in the
medium.4 The sixth coordination site is occupied by a NO (as
isolated) or an HxO molecule (after photolysis).8,9 In a recent
†
Department of Chemistry, Stanford University.
Bioengineering Laboratory, RIKEN.
⊥ Department of Chemistry, University of Washington.
‡ Stanford Synchrotron Radiation Laboratory, Stanford University.
§
(1) Endo, I.; Nojiri, M.; Tsujimura, M.; Nakasako, M.; Nagashima, S.; Yohda,
M.; Odaka, M. J. Inorg. Biochem. 2001, 83, 247-253.
(2) Yamada, H.; Kobayashi, M. Biosci., Biotechnol., Biochem. 1996, 60, 13911400.
(3) (a) Nagashima, S.; Nakasako, M.; Dohmae, N.; Tsujimura, M.; Takio, K.;
Odaka, M.; Yohda, M.; Kamiya, N.; Endo, I. Nat. Struct. Biol. 1998, 5,
347-351. (b) Huang, W.; Jia, J.; Cummings, J.; Nelson, M.; Schneider,
G.; Lindqvist, Y. Structure 1997, 5, 691-699.
(4) Nagasawa, T.; Takeuchi, K.; Yamada, H. Eur. J. Biochem. 1991, 196, 581589.
(5) Payne, M. S.; Wu, S.; Fallon, R. D.; Tudor, G.; Stieglitz, B.; Turner, I.
M.; Nelson, M. J. Biochemistry 1997, 36, 5447-5454.
(6) Miyanaga, A.; Fushinobu, S.; Ito, K.; Shoun, H. Eur. J. Biochem. 2004,
271, 429-438.
(7) Watanabe, N.; Nakayama, H.; Odaka, M.; Kawano, Y.; Takio, K.; Kamiya,
N.; Nagamune, T.; Endo, I. J. Inorg. Biochem. 2001, 86, 475-475.
Figure 1. Proposed active-site structure of NHase from the 1.7-Å resolution
crystal structure.3a
crystal structure of the inactive NO-bound form3a (Figure 1a),
the cysteine ligands are post-translationally modified to RSO(H) and RSO2(H) in contrast to a previously reported crystal
structure showing non-oxidized CysS- coordination (Figure 1).3b
However, aside from FT-IR, there are no direct spectroscopic
probes of the CysS- ligand oxidation states in the active form
of the enzyme.10 Although active sites having transition-metal(8) Odaka, M.; Fujii, K.; Hoshino, M.; Noguchi, T.; Tsujimura, M.; Nagashima,
S.; Yohda, M.; Nagamune, T.; Inoue, Y.; Endo, I. J. Am. Chem. Soc. 1997,
119, 3785-3791.
(9) Endo, I.; Odaka, M.; Yohda, M. Trends Biotechnol. 1999, 17, 244-248.
Submitted to Journal of the American Chemical Society
Work supported in part by Department of Energy contract DE-AC02-76SF00515
bound oxidized thiolate residues are rare in nature, mutational
studies on these thiolate residues have shown them to be
catalytically relevant.11,12
The catalytic mechanism of this site is not well understood,
and there are several proposals regarding the role of the lowspin FeIII center in activating nitriles for hydrolysis.13 The
possible functional role of these modified cysteines, and the
electronic structure of the as-isolated [FeNO]6 species (using
the Enemark-Feltham nomenclature, where the superscript 6
refers to the total number of valence electrons on the iron and
the π* orbitals of NO) and its observed photochemistry, have
been a focus of several recent experimental and theoretical
studies.14-18 A significant number of structurally relevant model
complexes have been reported, some of which bind nitriles and
some of which reversibly bind NO and show photolytic
behavior.19-22 Furthermore, the study of a series of model
complexes having oxidized ligands has shown that this oxidation
can significantly alter the pKa of a water bound to the low-spin
FeIII.23
Ligand K-edge X-ray absorption spectroscopy (XAS) is a
direct probe of a ligand’s chemical nature and its bonding to a
metal.24,25 The primary transition at the K-edge is 1s f 4p. In
the case of sulfur, transitions to other unoccupied antibonding
orbitals, including the C-S σ*, O-S σ*, and M3d-S antibonding orbitals, gain intensity due to sulfur 3p mixing. The observed
intensity of these transitions is then directly proportional to the
extent of this mixing.26 This ligand K-edge XAS method has
been used to investigate bonding in several models having
different types of chlorine- and sulfur-based ligands and in the
active sites of NiSOD and nickel dithiolenes, blue copper, CuA,
and iron-sulfur proteins.27-32 In those studies, where the focus
(10) Noguchi, T.; Nojiri, M.; Takei, K.; Odaka, M.; Kamiya, N. Biochemistry
2003, 42, 11642-11650.
(11) Murakami, T.; Nojiri, M.; Nakayama, H.; Odaka, M.; Yohda, M.; Dohmae,
N.; Takio, K.; Nagamune, T.; Endo, I. Protein Sci. 2000, 9, 1024-1030.
(12) Piersma, S. R.; Nojiri, M.; Tsujimura, M.; Noguchi, T.; Odaka, M.; Yohda,
M.; Inoue, Y.; Endo, I. J. Inorg. Biochem. 2000, 80, 283-288.
(13) Endo, I.; Nojiri, M.; Tsujimura, M.; Nakasako, M.; Nagashima, S.; Yohda,
M.; Odaka, M. J. Inorg. Biochem. 2001, 83, 247-253.
(14) Mascharak, P. K. Coord. Chem. ReV. 2002, 225, 201-214.
(15) Kobayashi, M.; Shimizu, S. Eur. J. Biochem. 1999, 261, 1-9.
(16) Boone, A. J.; Chang, C. H.; Greene, S. N.; Herz, T.; Richards, N. G. J.
Coord. Chem. ReV 2003, 238, 291-314.
(17) Harrop, T. C.; Mascharak, P. K. Acc. Chem. Res. 2004, 37, 253-260.
(18) Grapperhaus, C. A.; Patra, A. K.; Mashuta, M. S. Inorg. Chem. 2002, 41,
1039-1041.
(19) Kovacs, J. A. Chem. ReV. 2004, 104, 825-848.
(20) Shearer, J.; Kung, I. Y.; Lovell, S.; Kaminsky, W.; Kovacs, J. A. J. Am.
Chem. Soc. 2001, 123, 463-468.
(21) Shearer, J.; Jackson, H. L.; Schweitzer, D.; Rittenberg, D. K.; Leavy, T.
M.; Kaminsky, W.; Scarrow, R. C.; Kovacs, J. A. J. Am. Chem. Soc. 2002,
124, 11417-11428.
(22) Noveron, J. C.; Olmstead, M. M.; Mascharak, P. K. J. Am. Chem. Soc.
2001, 123, 3247-3259.
(23) Tyler, L. A.; Noveron, J. C.; Olmstead, M. M.; Mascharak, P. K. Inorg.
Chem. 2003, 42, 5751-5761.
(24) Solomon, E. I.; Hedman, B.; Hodgson, K. O.; Dey, A.; Szilagyi, R. K.
Coord. Chem. ReV. 2005, 249, 97-129.
(25) Glaser, T.; Hedman, B.; Hodgson, K. O.; Solomon, E. I. Acc. Chem. Res.
2000, 33, 859-868.
(26) Neese, F.; Hedman, B.; Hodgson, K. O.; Solomon, E. I. Inorg. Chem. 1999,
38, 4854-4860.
(27) Szilagyi, R. K.; Bryngelson, P. A.; Maroney, M. J.; Hedman, B.; Hodgson,
K. O.; Solomon, E. I. J. Am. Chem. Soc. 2004, 126, 3018-3019.
(28) George, S. D.; Metz, M.; Szilagyi, R. K.; Wang, H. X.; Cramer, S. P.; Lu,
Y.; Tolman, W. B.; Hedman, B.; Hodgson, K. O.; Solomon, E. I. J. Am.
Chem. Soc. 2001, 123, 5757-5767.
(29) Szilagyi, R. K.; Lim, B. S.; Glaser, T.; Holm, R. H.; Hedman, B.; Hodgson,
K. O.; Solomon, E. I. J. Am. Chem. Soc. 2003, 125, 9158-9169.
(30) Anxolabéhère-Mallart, E.; Glaser, T.; Frank, P.; Aliverti, A.; Zanetti, G.;
Hedman, B.; Hodgson, K. O.; Solomon, E. I. J. Am. Chem. Soc. 2001,
123, 5444-5452.
(31) Dey, A.; Glaser, T.; Moura, J. J. G.; Holm, R. H.; Hedman, B.; Hodgson,
K. O.; Solomon, E. I. J. Am. Chem. Soc. 2004, 126, 16868-16878.
was on the metal-sulfur bonding and its perturbation by the
protein environment, it was found that the S K-edge is very
sensitive to the Zeff of a ligand, the chemical environment of
the ligand, and the Zeff of the metal to which it is bound.33
In this study we apply XAS and density functional theory
(DFT) to a series of crystallographically characterized model
complexes to identify signature spectroscopic features associated
with thiolate-based ligands having different oxidation and
protonation states. We then use these features to identify the
ligands present in the active site of the protein NHase in its
NO-bound inactive and photolyzed active forms. We further
investigate the electronic structure of [FeNO]6 NHase using DFT
calculations in conjunction with the spectroscopic results.
Experimental Details
Sample Preparation. All four model complexes, FeIII(ADIT)2 (1),
FeIII(ADIT)(ADIT-O) (2), [CoIII((η2-SO)(SO2)N3(Pr,Pr))] (3), and FeIII(ADIT)(ADIT-O-ZnCl3) (4), were synthesized as reported in the
literature.19,34 For XAS experiments, the samples were ground into a
fine powder, dispersed as thinly as possible on sulfur-free Mylar tape
in a dry, anaerobic glovebox (N2) atmosphere, and mounted across the
window of an aluminum plate. This procedure has been verified to
minimize self-absorption effects. A 6.35-µm polypropylene film window
protected the solid samples from exposure to air during transfer from
the glovebox to the experimental sample chamber.
The NO-bound NHase protein was expressed and purified as
described in ref 35. The protein solutions (in 100 mM phosphate buffer,
pH 7.5) were pre-equilibrated in a water-saturated He atmosphere for
∼1 h to minimize bubble formation in the sample cell. The solution
was loaded via a syringe into a Pt-plated Al block sample holder, sealed
in front using a 6.3-µm polypropylene window. To ensure photolysis,
the protein sample was illuminated by a 400-W tungsten lamp for 30
min before the XAS data measurements.
Data Collection and Reduction. XAS data were measured at the
Stanford Synchrotron Radiation Laboratory using the 54-pole wiggler
beam line 6-2. Details of the experimental configuration for low-energy
studies have been described in an earlier publication.33 The data
reduction and error analysis follow the same method discussed
previously.36
Fitting Procedure. Pre-edge features were fit by pseudo-Voigt line
shapes (sums of Lorentzian and Gaussian functions). This line shape
is appropriate as the experimental features are expected to be a
convolution of a Lorentzian transition envelope and a Gaussian line
shape imposed by the spectrometer optics.37,38 A fixed 1:1 ratio of
Lorentzian to Gaussian contribution successfully reproduced the preedge features. The rising edge was also fit with pseudo-Voigt line
shapes. Good fits reproduce the data and its second derivative using a
minimum number of peaks. The intensity of a pre-edge feature (peak
area) is the sum of the intensity of all the pseudo-Voigt peaks which
successfully fit the feature in a given fit. The reported intensity values
for the proteins are averages of all good pre-edge fits.
Computational Details. All calculations were performed on dualCPU Pentium Xeon 2.8 GHz work stations using the Amsterdam
Density Functional (ADF) program, versions 2004.01, developed by
(32) Glaser, T.; Bertini, I.; Moura, J. J. G.; Hedman, B.; Hodgson, K. O.;
Solomon, E. I. J. Am. Chem. Soc. 2001, 123, 4859-4860.
(33) Hedman, B.; Frank, P.; Gheller, S. F.; Roe, A. L.; Newton, W. E.; Hodgson,
K. O. J. Am. Chem. Soc. 1988, 110, 3798-3805.
(34) Lugo-Mas, P.; Xu, L.; Davin, S. D.; Benedict, J.; Kaminsky, W.; Kovacs,
J. A. J. Am. Chem. Soc., submitted.
(35) Nojiri, M.; Yohda, M.; Odaka, M.; Matsushita, Y.; Tsujimura, M.; Yoshida,
T.; Dohmae, N.; Takio, K.; Endo, I. J. Biochem. 1999, 125, 696-704.
(36) Shadle, S. E.; Hedman, B.; Hodgson, K. O.; Solomon, E. I. Inorg. Chem.
1994, 33, 4235-4244.
(37) Agarwal, B. K. X-ray Spectroscopy; Springer-Verlag: Berlin, 1979; pp
276 ff.
(38) Tyson, T. A.; Roe, A. L.; Frank, P.; Hodgson, K. O.; Hedman, B. Phys.
ReV. B 1989, 39A, 6305-6315.
Figure 3. S K-edge XAS of free cysteine at pH 7 (black), OHCH2SO2(gray) and OHCH2SO2H (dashed gray), and model complexes 1 (blue), 2
(dashed blue), 3 (red), and 4 (green).
Figure 2. Schematic structures of the model complexes of NHase studied.
Baerends et al.39,40 A triple-ζ Slater-type orbital basis set (ADF basis
set TZP) with a single polarization function at the local density
approximation of Vosko, Wilk, and Nusair,41 with nonlocal gradient
corrections of Becke42 and Perdew,43 was employed. The molecular
orbitals were plotted using Molden version 5.1, and the Mulliken44
population analyses were performed using the AOMix45 program. The
calculations of the protein active site were performed using the BP86
functional as well as the hybrid B3LYP46 functional with the Gaussian
03 package.47 The solvation calculations (single points and optimizations) were performed using the PCM48 method and epsilon of 4.0.
The optimizations were performed with a mixed basis set with 6-311g*
on Fe, S, N, and O and 6-31g* on C and H. The single-point calculations
were performed with a 6-311+g** basis set on all atoms.
Results and Analysis
A. S K-Edge XAS of Model Complexes and Free Ligands.
The X-ray absorption spectra of the structurally characterized
model complexes [FeIII(ADIT)2]+ (1), [FeIII(ADIT)(ADIT-O)]+
(2), [CoIII(η2-SO)(SO2)N3(Pr,Pr)]+ (3), and [FeIII(ADIT)(ADITO-ZnCl3)]+ (4) (Figure 2) were used as references for ligand
oxidation state determination for the NHase enzyme (Figure 1).49
Complex 1 contains a low-spin FeIII atom coordinated by two
thiolates, one of which is oxidized in the low-spin complex (2).
Complex 4 has a ZnCl3 moiety coordinated to the S-O moiety
in 2, while 3 has a low-spin CoIII atom ligated by an
η2-coordinated S-O- moiety and an RSO2 moiety coordinated
through sulfur, as in the proposed active site of NHase (Figure
1).3
The S K-edge XAS of free cysteine at pH 7.0 (black line in
Figure 3) has an intense RS-1s f RS-C-Sσ* transition at 2473.0
eV. The spectra of aqueous solutions of sulfinic acid at pH 12.0
(Figure 3, solid gray, deprotonated) and pH 1.3 (Figure 3, dashed
(39)
(40)
(41)
(42)
(43)
(44)
(45)
(46)
(47)
(48)
(49)
Baerends, E. J.; Ellis, D. E.; Roos, P. Chem. Phys. 1973, 2, 41-51.
te Velde, G.; Baerends, E. J. Int. J. Comput. Phys. 1992, 99, 84-98.
Vosko, S. H.; Wilk, L.; Nusair, M. Can. J. Phys. 1980, 58, 1200-1211.
Becke, A. D. Phys. ReV. A: Gen. Phys. 1988, 38, 3098-3100.
Perdew, J. P. Phys. ReV. B 1986, 33, 8822-8224.
Mulliken, R. S. J. Chem. Phys. 1955, 23, 1833-1840.
Gorelsky, S. I. AOMix Program rev.6.04 (http://www.sg-chem.net). Gorelsky, S. I.; Lever, A. B. P. J. Organomet. Chem. 2001, 635, 187-196.
Becke, A. D. J. Chem. Phys. 1993, 98, 5648-5652.
Frisch, M. J.; et al. Gaussian 03, Revision C.02; Gaussian, Inc.: Wallingford
CT, 2004.
Miertus, S.; Scrocco, E.; Tomasi, J. Chem. Phys. 1981 55, 117-129.
The S K-edge XAS of free ligands having varying oxidation states is given
in Figure S1 in the Supporting Information for reference. These results
were published in ref 33.
gray, protonated) are almost identical, and the RSO2-1s f
RSO2-C-Sσ* and RSO2-1s f RSO2-O-Sσ* transitions show up
as a strong absorption feature at 2477.0 eV. The shift of this
feature from 2473 eV in cysteine to 2477 eV in sulfinic acid
reflects the stabilization of the S1s orbital due to the higher
oxidation state of sulfur in the latter.33,50 The S K-edge XAS of
complex 1 (Figure 3, solid blue) shows two pre-edge transitions
around 2470 eV (2469.8 and 2470.5 eV) and a transition at
2473.1 eV, corresponding to RS-1s f Fe3d and RS-1s f
RS-C-Sσ* transitions, respectively. The spectrum of complex 2
(Figure 3, dashed blue) also shows the two pre-edge transitions
from the thiolate (now well separated) at 2469.8 and 2470.9
eV and an RS-1s f RS-C-Sσ* transition at 2473.3 eV, as well
as a new third broad transition with a maximum at 2475.3 eV,
which is now assigned to an envelope of the RSO-1s f
RSO-C-Sσ* and RSO-1s f RSO-O-Sσ* transitions. These
transitions are shifted higher in energy relative to the RS-1s f
RS-C-Sσ* transitions due to the deeper energy of the 1s orbital
of the oxidized sulfur in RSO-. Note that the RSO-1s f Fe3d
pre-edge transition should also have intensity due to covalent
bonding of the RSO- ligand to the metal d-orbitals through the
sulfur. However, this would largely overlap the RS-1s f
RS-C-Sσ* transition at 2473.1 eV in 2. This transition is, in fact,
clearly observed at 2473.2 eV for 3 (Figure 3, red), a complex
in which there is no RS- ligand and hence no RS-1s f RS-C-Sσ*
transition. As for 2, complex 3 also exhibits RSO-1s f
RSO-C-Sσ* and RSO-1s f RSO-O-Sσ* transitions at ∼2475 eV
(maxima at 2474.9 and 2475.9 eV). There is an additional
higher-energy feature in the spectrum of 3 at 2478.8 eV, which
is assigned to the RSO2-1s f RSO2-C-Sσ* and RSO2-1s f
RSO2-O-Sσ* transitions. Note that this transition envelope is
shifted up in energy by almost 2 eV for complex 3 (2478.8 eV,
Figure 3, red) relative to free sulfinic acid (2477.0 eV, Figure
3, solid gray). This shift has contributions from both the increase
in Zeff of sulfur in RSO2- due to its covalent bonding to the
CoIII ion, which shifts the sulfur 1s orbital to deeper energy,
and from the shorter S-O bonds in 3 (1.45 Å) compared to the
those in the free ligand (1.51 Å), which shifts the σ* orbital to
higher energy (Table 1). The XAS of complex 4 (Figure 3,
green) is very similar to that of 2 (Figure 3, dashed blue) and
has the same assignment of features, i.e., RS-1s f Fe3d and
(50) XAS data for a stable uncoordinated RSO- species were unavailable, so
we used the model complex data to identify the spectroscopic feature of
this ligand.
Table 1. DFT-Optimized Geometric Parametersa
RS-
FeIII(ADIT)2 (1)
FeIII(ADIT)(ADIT-O) (2)
CoIII((η2-SO)(SO2)N3 (Pr,Pr)) (3)
FeIII(ADIT)(ADIT-O-ZnCl3) (4)
CH3SOCH3SOH
CH3SO-- -C(NH2)3+
CH3SO2CH3SO2H
a
RSO(H)
M−S
C−S
2.21 (2.20)
2.19 (2.17)
1.84 (1.82)
1.85 (1.84)
2.20 (2.16)
1.84 (1.84)
RSO2(H)
M−S
C−S
S−O
2.31 (2.25)
2.18 (2.13)
2.29 (2.25)
1.87 (1.87)
1.85 (1.83)
1.86 (1.87)
1.86
1.80
1.82
1.55 (1.55)
1.57 (1.55)
1.56 (1.57)
1.60
1.72
1.64
M−S
C−S
S−O
2.18 (2.12)
1.87 (1.86)
1.47 (1.46)
1.90
1.81
1.53
1.49, 1.69
Crystal structure parameters are given within parentheses.19
RS-1s f RS-C-Sσ* transitions around 2470 and 2473 eV,
respectively, and RSO-1s f RSO-C-Sσ* and RSO-1s f
RSO-O-Sσ* transitions at 2474.9 and 2475.9 eV, respectively.
It is also important to note that the broad, higher-energy
transition at ∼2475 eV in 2, associated with the RSO-1s f
RSO-C-Sσ* and RSO-1s f RSO-O-Sσ*, is partly split in 3 and
is clearly split into two features, at 2474.9 and 2475.9 eV, in 4.
Note that complexes 3 and 4 both have the oxygen atom of the
RSO- coordinated to a metal (CoIII in 3 and ZnII in 4), and this
interaction appears to energy-split the broad feature at ∼2475
eV. DFT calculations were used to evaluate the effects of metal
coordination to the S, protonation/metal coordination to the O
of RSO-, and protonation of RSO2-.
B. DFT Calculations of the Model Complexes and Ligands.
Geometry-optimized DFT calculations were performed for
complexes 2 and 3 and for the free ligands CH3SO-, CH3SOH,
CH3SO2-, and CH3SO2H. The optimized bond lengths are in
overall good agreement with published crystal structures (Table
1). The optimized structures are used to obtain ground-state S3p
orbital distributions in the unoccupied virtual orbitals for these
complexes and free ligands. These calculations were used to
estimate the relative energies (zero defined as 2390 eV) and
intensities of their S K-edge XAS (Figure 4, predicted S
K-edge).51 Though the absolute transition energies were underestimated (due to lack of relativistic corrections), the relative
energies of the pre-edge and the rising edge features are in good
agreement with the experimental spectra in Figure 3.
The intensity distribution for 2 (Figure 4, dashed blue) shows
two features corresponding to RS-1s f Fe3d and RS-1s f
RS-C-Sσ* transitions at 4.0 eV (first transition split due to ligand
field effects on the 3d orbitals) and 7.0 eV, respectively. It also
shows a third feature at 9.0 eV which corresponds to contributions of the RSO-1s f RSO-C-Sσ* and RSO-1s f RSO-O-Sσ*
transitions. Calculations for complex 3 give the RSO-1s f Co3d
transition at 6.3 eV and the RSO-1s f RSO-C-Sσ* and
RSO-1s f RSO-O-Sσ* transitions at 9.0 eV (Figure 4, red). Note
that the σ* transitions are separated in energy by 1 eV and that
the calculations reproduce the experimentally observed energy
splitting of this feature. There is an additional higher-energy
feature for 3 (Figure 4, red), corresponding to the overlapping
contributions from RSO2-1s f RSO2-C-Sσ* and RSO2-1s f
RSO2-O-Sσ* transitions. Calculations on both free CH3SO2- and
CH3SO2H show this feature at 9-11 eV (Figure 4, solid gray
and dashed gray, respectively). These calculations reproduce
the 2-eV increase in energy of this feature observed in the data
for 3 relative to that of the free ligand and show no significant
effect of protonation of the oxygen of the RSO2- group on the
S3p distribution, consistent with experiment (Figure 3, solid gray
and dashed gray). A calculation performed on the CH3SO2fragment, having the geometry of complex 3 (i.e., with shorter S-O bond lengths) but not coordinated to a metal ion,
shows no significant shift of the RSO2-1s f RSO2-C-Sσ* and
RSO2-1s f RSO2-O-Sσ* transition energies, indicating that the
experimentally observed 2-eV shift of this feature between the
free ligand and complex 3 (Figure 3, red and gray lines) is a
result of CoIII coordination to the sulfur of RSO2-. This shifts
charge density from S to CoIII, stabilizing the S 1s orbital energy
in complex 3 relative to that of the free ligand.
A DFT calculation on CH3SO- shows one feature at 7.0 eV
that corresponds to an envelope of the RSO-1s f RSO-C-Sσ*
and RSO-1s f RSO-O-Sσ* transitions (Figure 4, green).52 In
contrast to calculations on RSO2(H), the DFT calculation on
the free CH3SOH ligand shows dramatic differences in the S3p
energy/intensity distribution relative to that of the CH3SOligand. The RSO-1s f RSO-C-Sσ* and RSO-1s f RSO-O-Sσ*
transitions, which combine into one peak at 7.0 eV in the
deprotonated ligand (Figure 4, solid green), are now split into
two peaks (Figure 4, dashed green) at 6.0 eV (the RSO-1s f
RSO-O-Sσ* transition) and 8.0 eV (the RSO-1s f RSO-C-Sσ*
transition) upon protonation. Geometry-optimized DFT calculations on these free ligands (Table 1) show that the C-S bond
(51) The calculated S3p characters were scaled up by an approximate factor of
2 for every two units of change in its oxidation number to account for the
increase in Zeff, which will increase the transition dipole integral, i.e.,
〈S1s|r|S3p〉, leading to an increase in absorption intensity.
(52) This transition is not shifted in energy in the CH3SO- fragment, having
the geometric parameters of complex 2, but is shifted (by 1.6 eV) to 8.9
eV in 2, reflecting the shift of charge from the coordinated sulfur atom
due to covalent interaction, as also observed in complex 3.
Figure 4. DFT-calculated ground-state S3p mixing in 2 (dashed blue), 3
(red), CH3SO2- (gray), CH3SO2H (dashed gray), CH3SO- (green), and CH3SOH (dashed green). Zero defined at 2390 eV.
Figure 5. S K-edge XAS of NHase-NO (black), NHase-photoactivated
(dashed black), simulation with 1 only (blue), and simulation with 3 (red).
Inset: Expanded pre-edge region of NHase-NO and NHase-photoactivated.
Figure 6. Second derivative of XAS data of 2 (dashed blue), 3 (red), 4
(green), NHase-NO (black), and NHase-photoactivated (dashed black).
shortens by 0.06 Å and the S-O bond elongates by 0.1 Å on
protonation of CH3SO-. This shifts the C-S σ* orbital higher
and the O-S σ* orbital lower in energy, resulting in the
observed energy splitting of these features. A similar elongation
of the S-O and shortening of the C-S bonds is observed in
complexes 4 and 3 (O-S 1.56 Å, C-S 1.86 Å and O-S 1.57
Å, C-S 1.87 Å, respectively) relative to 2 (O-S 1.55 Å, C-S
1.85 Å).53 Thus, the experimentally observed energy splitting
of the 2474-2476 eV features of complexes 3 and 4 is due to
the weakening of the S-O and strengthening of the C-S bonds
as a result of the metal coordination to the RSO- oxygen atom.
The larger energy splitting for complex 4 is probably due to its
stronger coordination to ZnII. As shown in Figure 4, full
protonation produces the largest effect. It should be noted that
for RSO2(H) the changes in bond lengths with protonation are
smaller (1.53 Å vs 1.49 and 1.69 Å) and the split of the feature
at 9-11 eV does not change significantly.
In summary, from the model studies it is found that the
S1s f C-S σ* and S-O σ* transitions are very sensitive to
the Zeff of the sulfur. RS- has RS-1s f Fe3d and RS-1s f
RS-C-Sσ* transitions at ∼2470 and ∼2473 eV, respectively,
RSO- has RSO-1s f RSO-C-Sσ* and RSO-1s f RSO-O-Sσ*
transitions at 2475 eV which split by 2 eV on protonation, and
RSO2- has the RSO2-1s f RSO2-C-Sσ* and RSO2-1s f
RSO2-O-Sσ* transitions at 2477-2479 eV (depending on metal
ion coordination) which are not significantly affected by
protonation.
C. S K-Edge XAS of Nitrile Hydratase. The S K-edge XAS
of the inactive NO-bound form of NHase shows four distinct
features (Figure 5, black). The lowest-energy feature, at 2470.1
eV (inset), is assigned as the RS-1s f Fe3d transition. The broad
envelope at 2472-2474 eV can be assigned as a composite of
RS-1s f RS-C-Sσ* transitions of the ligated cysteine, two free
cysteines and nine methionines. The other higher-energy
features, at 2476.1 and 2478.5 eV, are relatively weak; however,
a simulated spectrum generated by the normalized addition of
the spectra of aqueous methionine and cysteine at pH 7.5 and
1, representing thiolate coordination but no oxidized sulfur
(Figure 5, blue), shows no higher-energy features. Alternatively,
simulation including the oxidized ligands (Figure 5, red,
complex 3) shows two higher-energy features at 2475-2476
and 2475.5-2479.5 eV, similar to those for NHase-NO at
2476.1 and 2478.5 eV, indicating that they are associated with
the oxidized sulfur ligands present in the NO-bound form of
the protein. The feature at 2478.5 eV for the enzyme can be
assigned to RSO2-. Note that this is at a higher energy than for
the RSO2-1s f RSO2-C-Sσ* and RSO2-1s f RSO2-O-Sσ*
transitions observed for the free RSO2- ligand (at 2476-2478
eV, Figure 3, gray) but lower than those for the CoIII complex
3 (at 2478.8 eV, Figure 3, red), indicating coordination of the
CysSO2- ligand at the protein active site but with a weaker
bond relative to complex 3.
The feature at 2476.1 eV could originate from RSO-,
although this feature is shifted to higher energy by ∼1 eV than
is observed for RSO- in complexes 2 and 3. In sections A and
B (vide supra), it is shown that the energy of the RSO- feature
is sensitive to the chemical environment of the oxygen.
Inspection of the second derivatives of the absorption data
(where minima in second derivative represent maxima in the
absorption spectrum) (Figure 6) reveals that the NHase-NO
enzyme data has a minimum at 2476.1 eV (Figure 6, black),
while the RSO--ligated complex (2) has a broad minimum at
2475.2 eV (Figure 6, dashed blue). In 4 (Figure 6, green), where
Zn2+ is strongly bound to the O of RSO-, the RSO-1s f
RSO-S-Oσ* feature has shifted down to 2474.8 eV, reflecting a
weaker S-O bond in 4, while the RSO-1s f RSO-C-Sσ*
negative feature remains at 2476.1 eV. As found in section B,
protonation of RSO- will shift the S-O σ* transition to 2 eV
lower in energy than the C-S σ* transition, due to the
elongation of the S-O bond. Hence, the absence of a single
minimum in the second derivative, corresponding to a peak in
absorption between 2474 and 2475 eV in the NHase data, argues
against the presence of an RSO- residue and indicates that the
cysteine sulfenic acid residue in the NHase active site is
protonated. While the RSO(H)1s f RSO(H)C-Sσ* transition is
observed in the data for NHase (Figure 6, black) at 2476.1 eV,
about 1.0 eV higher than the single peak observed for 2 (which
can be used as an estimate of the energy position of a pure
RSO- feature), the RSO(H)1s f RSO(H)S-Oσ* transition of
Nhase, which is estimated to be 2 eV lower in energy relative
to the RSO(H)1s f RSO(H)C-Sσ* transition, could be merged
into the RS-1s f RS-C-Sσ* transition envelope at 2472-2474
eV.54
(53) The optimized S-O bond in 2 is 0.04 Å longer than in the crystal structure,
while those for 3 and 4 are within 0.01 Å (Table 1).
(54) Note that there is a minimum in the protein XAS data at 2474 eV, which
may originate from RSO-1s f RSO-S-Oσ*. However, in the presence of
nine methionine residues this feature is not observed in the absorption data.
Figure 7. (A) 1.6-Å resolution crystal structure of the NHase-NO complex active site. (B) Optimized geometry, gas phase. (C) Optimized geometry, PCM,
) 4.0.
S K-edge XAS data for the photoactivated protein (Figure 5,
dashed black) show no significant change relative to the data
for the inactive NO-bound NHase. This indicates that the
oxidation states of the metal and the sulfur ligands and the
protonation state of RSO- are virtually identical in the NObound inactive and the photolyzed active enzyme. However,
there is a markedly increased pre-edge intensity upon photolysis
(Figure 5, inset), which is analyzed in section E.
D. DFT Calculations on the Active Site of NHase. While
the S K-edge XAS data for the protein active site and model
complexes enabled us to assign the chemical nature of two of
the three cysteinyl ligands (CysS- and CysSOH), it could not
help to discern the protonation state of the CysSO2- ligand.
Geometry-optimized DFT calculations were performed on the
crystallographically characterized NO-bound inactive form to
evaluate this issue (Figure 7A). The active site was modeled
using two oxidized cysteines coordinated to the Fe in the
equatorial plane (Cys 114, Cys 112) and a methyl thiolate
modeling the axial cysteine (Cys 109). There are two hydrogen
bonds from nearby arginine residues (Arg 141 and Arg 56)
which are modeled by two C(NH2)3+ fragments.55 The optimized
structures show that the CysSO2- remains in its ionized form.
(55) The central C of the C(NH2)3+ species was frozen during optimization.
Table 2. Pre-edge Analysis Results for NHase Data
Fe orbital
e
t2
energy (eV)
NHase-NO
NHase-photoactivated
2469.6
% S3p
energy (eV)
% S3p
9
2470.1
2470.2
20
15
This is reasonable as the pKa of RSO2- is 6-7 and that of
arginine is 12-14; hence, they are expected to be ionized under
the experimental conditions. Also, in a vacuum the charged
species should neutralize while they may not do so in a PCM
calculation. On the contrary, the proton from the C(NH2)3+
fragment, H-bonding to CysSO-, shifts to the CysSO- oxygen,
producing a neutral CysSOH species (Figure 7B). This computational model agrees well with the geometric structure
obtained from the XAS data as the optimized geometry displays
a long (1.68 Å) S-O bond upon protonation, which will split
the RSO-1s f RSO-C-Sσ* and RSO-1s f RSO-O-Sσ*, as
observed experimentally. However, it must be noted that the
pKa of the RSO- residue is 10-11 and that for arginine is 1214.56 As indicated above, the protonation state of the arginineCysSO unit will depend on the dielectric constant of the
medium. Geometry optimizations were also performed using a
PCM solvation model with an epsilon of 4.0. The results of
this optimization show that the CysSO- and the C(NH2)3+
residues remain charge-separated after optimization (Figure 7C),
in contrast to the vacuum-based calculations and the experimental results from XAS. However, the energy difference
between the CysSOH-C(NH2)2(NH) and the CysSO-C(NH2)3+ forms is only 4 kcal/mol and depends on modeling
of the active site and the functional used (4 kcal/mol for B3LYP
and 7 kcal/mol for BP86).57 This energy difference corresponds
to a pKa difference of 3 units. An electrostatic calculation (not
included in the DFT modeling) of the pKa’s for the buried
arginines in Nhase shows that these vary over the range 16.6
(the surface residues have ∼12) to 8.5, with one of the two
(Arg 167) H-bonded to the SO- ligand having a pKa of 9.3.58
E. Pre-edge Analysis. Due to covalent interaction with the
axial CysS-, the unoccupied 3d orbitals of the NO-bound NHase
active site will have S3p mixing, and hence will exhibit
RS-1s f Fe3d transition intensity, which is proportional to the
coefficient of S3p mixing in these orbitals. The S K-edge XAS
of the NO-bound inactive form has a distinct pre-edge feature
with a maximum at 2470.0 eV (Figure 6, inset, black). The
renormalized intensity of this pre-edge feature is 0.69 unit, which
correspond to 20% S3p character mixed into the antibonding 3d
orbital (Table 2). The NHase-photoactivated sample has two
pre-edge features, one at 2469.6 eV and a second at 2470.2
eV, with 0.31 and 0.51 unit of intensity, respectively (Figure 6,
inset, dashed black). This indicates that the active site of the
photolyzed form has two 3d antibonding orbitals with 9% and
15% S3p character (Table 2).
(56) Okuyama, T.; Miyake, K.; Fueno, T.; Yoshimura, T.; Soga, S.; Tsukurimichi, E. Heteroat. Chem. 1992, 3, 577-583.
(57) Given the resolution of the crystal structure, it is not possible to distinguish
between calculated structures B ( ) 0) and C ( ) 4.0) using structural
parameters.
(58) Estimated using the crystal structure of NO bound Nhase (pdb id 2AHJ)
with the software PROPKA: Li, H.; Robertson, A. D.; Jensen, Jan H.
Proteins 2005, 61, 704-721.
The MO diagrams of the NO-bound ([FeNO]6) and photolyzed NHase are given in Figure 8 (the β unoccupied orbitals).
Figure 8 (right) shows that the NO-bound form has a formal t26
configuration. There are two unoccupied NO π* LUMOs and
two higher-energy unoccupied e orbitals, dz2 and dx2-y2. The dz2
orbital (highlighted in blue) has 19% S3p character due to
pseudo-σ (S3p orbital slightly tilted off the Fe-S bond as the
angle C-S-Fe > 90°) overlap with the thiolate, which would
provide the intensity for a single pre-edge feature, as observed
in Figure 6 (inset) at 2470.1 eV. Photolysis cleaves the FeNO bond and creates a low-spin FeIII center with a H2O/OHmolecule coordinated in place of NO. Here the t2 hole is oriented
by the strong axial CysS- donor into the d-π orbitals (shaded
in red). This covalent mixing of the CysS- π into the d orbital
leads to the new lower-energy feature at 2469.5 eV in the preedge of the photolyzed protein. The higher-energy e feature
remains at 2470.2 eV, as the pseudo-σ overlap with this axial
CysS- ligand does not change.
The pre-edge region of the S K-edge XAS data is affected
by the chemical nature of the ligand, the ligand field of the
metal, and its Zeff. Here we use the pre-edge to probe the Zeff of
the metal ion and thus obtain insight into its effective oxidation
state. It is important to note that FeII complexes do not show a
pre-edge transition, as the Zeff for the FeII is low and this shifts
the Fe3d manifold of orbitals to higher energy, such that the
RS-1s f Fe3d transitions overlap with the RS-1s f RS-C-Sσ*
transition and are not distinguishable. The fit to the NHase data
shows a small pre-edge feature at 2470.1 eV (Figure 5 inset,
Table 2), which suggests that the Zeff of the Fe in the [FeNO]6
active site of NHase is significantly higher than that for lowspin FeII complexes59 and similar to that of FeIII NHase.
Discussion
S K-edge XAS is very sensitive to the chemical nature of
the sulfur atom. In this study we have used structurally
characterized synthetic model complexes to identify signature
transitions corresponding to different types of sulfur ligands.
Not only does the edge show dramatic differences upon
oxidation; in the case of RSO- the edge is also sensitive to
protonation. The data for both the NO-bound inactive and
photolyzed active forms of the NHase enzyme show the
presence of three types of sulfur ligands at the active site:
CysS-, CysSOH, and CysSO2(H). Although we could not
identify the protonation state of the CysSO2- using our data,
DFT calculations in both the gas phase and PCM show that it
is ionized (Figure 9). This model can now be used to understand
the role of these oxidized cysteines in catalysis.
The oxidation of the cysteine ligands has been proposed to
be catalytically relevant, either by affecting the binding of a
nitrile, followed by a nucleophilic attack by H2O, or by affecting
the binding of a H2O to the open coordination site of the Fe,
which then ionizes to release a H+ that catalyzes the hydrolysis.
A geometry-optimized DFT calculation on a low-spin FeIII
NHase active site with three thiolates (Figure 10) and using
H2O as the sixth ligand shows weak binding (Fe-OH2 ) 3.4
(59) The absence of a pre-edge feature in FeII sites has been studied in complexes
with weak field ligands (Cl-, RS-). Here, the 3d manifold in the NHaseNO complex will be further shifted to higher energy by the strong amide
and NO donors. Hence, the pre-edge transition will shift deeper into the
RS1s f RSC-Sσ* transitions of the rising edge. Thus, the spin state cannot
account for the low pre-edge energy.
Figure 8. DFT-calculated energy level diagram (β MO’s) of the NHase-NO active site (left) and NHase-photoactivated (right) (occupied orbitals are filled
diamonds and unoccupied orbitals are open diamonds). The MO’s having S3p mixing will have pre-edge transition intensity.
Figure 9. Active-site model of NHase NO-bound form developed from S
K-edge XAS and DFT calculations.
The electronic structure of linear [FeNO]6 units has been a
major focus of research in bio-inorganic chemistry.60,61 Münck
and co-workers, using Mössbauer spectroscopy, suggested that
the electronic structure of the NO-bound NHase can be described
as Fe(IV)-NO-.62 That conclusion was based on small isomer
shifts (0.03-0.05 mm/s) and high quadrupole splittings (1.32.0 mm/s) observed for an [FeNO]6 model.63 However, the
electronic properties of these complexes, investigated by recent
DFT calculations, have suggested that this unit can be described
as a low-spin Fe2+ coordinated to a NO+ unit.60,64 On photolysis,
a low-spin FeIII center is produced which is well-characterized
by EPR, Mössbauer, and resonance Raman spectroscopy.1,6 Note
that, from the XAS near-edge transitions, we know that the
chemical nature of the CysS- does not change upon photolysis,
and hence it serves as a spectator of the change in Zeff of the Fe
ion in this process. The pre-edge of this photolyzed ferric active
Å) affinity. However, calculations with the oxidized ligand set
show favorable H2O binding in this vacant coordination site
(Fe-OH2 ) 2.1 Å). Thus, the oxidized sulfur ligands are weaker
donors, which will increase the Lewis acidity of the FeIII center
and thus tune the ligand binding affinity to this vacant,
catalytically relevant, exchangeable coordination site.
(60) Serres, R. G.; Grapperhaus, C. A.; Bothe, E.; Bill, E.; Weyhermüller, T.;
Neese, F.; Wieghardt, K. J. Am. Chem. Soc. 2004, 126, 5138-5153.
(61) Tomita, T.; Haruta, N.; Aki, M.; Kitagawa, T.; Ikeda-Saito, M. J. Am. Chem.
Soc. 2001, 123, 2666-2667.
(62) Popescu, V.-C.; Münck, E.; Fox, B. G.; Sanakis, Y.; Cummings, J. G.;
Turner, I. M., Jr.; Nelson, M. J. Biochemistry 2001, 40, 7984-7991.
(63) Hauser, C.; Glaser, T.; Bill, E.; Weyhermuller, T.; Wieghardt, K. J. Am.
Chem. Soc. 2000, 122, 4352-4365.
(64) Greene, S. N.; Richards, N. G. J. Inorg. Chem. 2004, 43, 7030-7041.
Figure 10. (Left) H2O binding at a hypothetical NHase site where cysteines are not oxidized; Fe-O ) 3.4 Å. (Right) H2O binding at the actual enzymatic
site (Fe-O ) 2.1 Å).
site is observed at 2469-2470 eV (Figure 6, inset, dashed
black), an energy very similar to that of the pre-edge of the
inactive NO-bound form of NHase. This suggests that the Zeff
of the Fe atom in the [FeNO]6 NHase active site is very similar
to that of FeIII NHase rather than to that of FeII or FeIV. Also
note that the second derivative of the NO-bound form (Figure
6 inset, black) has only one feature at 2470.1 eV, corresponding
to one RS-1s f Fe3d (e) transition, while the second derivative
of the photolyzed protein has an additional feature in the preedge at 2469.5 eV, corresponding to the creation of a new lowenergy t2 hole in forming the low-spin FeIII center. This suggests
that the [FeNO]6 unit of inactive NHase has a Zeff similar to
that of FeIII but does not have a t2 hole. Such an electronic
structure would be consistent with strong back-donation from
the low-spin t26 iron to NO π* orbitals. From Figure 8, the
inactive NO-bound form has a low-spin t26 iron center that has
28-31% back-bonding interaction with the NO (3d character
summed over two unoccupied NO π* orbitals), which shifts
charge density from the formally FeII and increases its Zeff.
Acknowledgment. This research was supported by NIH
Grants NIH GM-40392 (E.I.S.), RR-01209 (K.O.H.), and
GM45881 (J.A.K.) and by the Bioarchitect Research Program,
RIKEN (M.O.). P.L.-M. gratefully acknowledges support from
NIH Predoctoral Minority Fellowship F31-GM073583-01. SSRL
operations are supported by the Department of Energy, Office
of Basic Energy Sciences. The SSRL Structural Molecular
Biology Program is supported by the National Institutes of
Health, National Center for Research Resources, Biomedical
Technology Program, and by the Department of Energy, Office
of Biological and Environmental Research.
Supporting Information Available: Complete ref 47; optimized coordinates of complexes 1-4, free ligands, and the NO
active site of NHase (in gas phase and using the PCM model).
This material is available free of charge via the Internet at
http://pubs.acs.org.
JA0549695